While most polypeptides of interest, e.g. pharmaceutically useful proteins, originate from eukaryotes, they are, due to high expression rates and high yields, usually produced in bacterial cells. However, the mechanism of polypeptide synthesis in bacteria differs from that in eukaryotes; polypeptides expressed in bacterial cells usually have either an additional foreign amino acid at the N-terminus or are inhomogenous in respect to their N-terminus, since cleavage of the additional amino acid can occur but remains incomplete most of the time.
Such inhomogeneity is however unacceptable in particular in the pharmaceutical field, because these polypeptides show properties that are different from the properties of the naturally occurring polypeptide, e.g. induction of antibody formation, half-life, pharmacokinetics etc. An N-terminus that deviates from the naturally occurring protein and/or is inhomogenous is an unacceptable feature. For the production of pharmaceutical polypeptides it is in most cases necessary to produce a nature-identical product (homogeneous with the correct N-terminus, which has no additional amino acids. The known methods attempt to reach this goal by incorporating additional steps in the process of polypeptide production, with expenditure of costs and materials, making further work up, the so-called downstream processing of the product, more complex.
Known methods for the production of a polypeptide in bacterial cells with a defined, homogenous N-terminus employ a fusion polypeptide comprising the polypeptide of interest and, N-terminally linked thereto, a polypeptide with autoproteolytic activity, preferably the autoprotease Npro of pestivirus. The autoproteolytic activity of the fusion partner leads to the cleavage of the polypeptide of interest with a homogenous N-terminus.
If a polypeptide is produced in the cytoplasm of bacterial cells, under certain conditions, the production rate of the polypeptide is faster than the folding kinetics. Therefore high density polypeptide aggregates are formed, which are deposited in the cytoplasm of the cell as inclusion bodies. The production of polypeptides in the form of inclusion bodies is of special interest for production on industrial scale, since the expressed polypeptide is present in the inclusion bodies in high amounts and a high degree of purity. Also, the inclusion bodies of the cell lack proteases, so that the polypeptide is protected when stored in inclusion bodies. In addition, inclusion bodies are easy to isolate. However, major drawbacks of production of polypeptides in the form of cytoplasmatic inclusion bodies are low solubility of the inclusion bodies and the necessity to refold the polypeptide.
Accordingly, processing of inclusion bodies is complex, especially since correct refolding is required in order to gain the biologically active form of the polypeptide of interest. Therefore, although the use of the autoproteolytic activity of a fusion polypeptide as described above consistently leads to the production of a polypeptide with a homogenous N-terminus, the process of purification of the desired product remains tedious, especially if it is expressed in form of cytoplasmatic inclusion bodies. The processing involves numerous steps including washing, refolding, cleavage, purification, and isolation.
Thus, the complex downstream processing poses a big challenge with regard to fast and cost effective production of polypeptides. This is exceedingly the case for production on industrial scale. Accordingly, there is an ongoing need for a simple and feasible process for production and purification of polypeptides.